Loss of biased signaling at a G protein-coupled receptor in overexpressed systems

G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β-arrestins. [Sarcosine1Ile4Ile8]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), fails to activate G protein in physiologically relevant models. Despite this, SII and several derivatives induce cellular signaling outcomes through β-arrestin-2-dependent mechanisms. However, studies reliant on exogenous AT1R overexpression indicate that SII is a partial agonist for G protein signaling and lacks β-arrestin-exclusive functional specificity. We investigated this apparent discrepancy by profiling changes in functional specificity at increasing expression levels of AT1R using a stably integrated tetracycline-titratable expression system stimulated with AngII, SII, and four other AngII analogs displaying different signaling biases. Unbiased and G protein-biased ligands activated dose-dependent calcium responses at all tested receptor concentrations. In contrast, β-arrestin-biased ligands induced dose-dependent calcium signaling only at higher AT1R overexpression levels. Using inhibitors of G proteins, we demonstrated that both Gi and Gq/11 mediated overexpression-dependent calcium signaling by β-arrestin-biased ligands. Regarding β-arrestin-mediated cellular events, the β-arrestin-biased ligand TRV026 induced receptor internalization at low physiological receptor levels insufficient for it to initiate calcium signaling. In contrast, unbiased AngII exhibited no relative preference between these outcomes under such low receptor conditions. However, with high receptor overexpression, TRV026 lost its functional selectivity. These results suggest receptor overexpression misleadingly distorts the bias of AT1R ligands and highlight the risks of using overexpressed systems to infer the signaling bias of GPCR ligands in physiologically relevant contexts.


Introduction
GPCRs constitute the largest family of cell surface receptors in the mammalian genome and, owing to their regulation of a wide range of physiological processes, are the most common target of therapeutic drugs [1,2]. Classically, GPCRs signal by activating heterotrimeric G proteins, which subsequently engage effector enzymes that modulate the production of second messengers [2]. In this paradigm, β-arrestins are recruited to activated and phosphorylated receptors and mediate desensitization by sterically hindering G protein coupling [3,4] and serving as adaptors for receptor endocytosis [5]. However, β-arrestins can also interact with many signaling proteins, thereby acting as transducers and initiating β-arrestin-dependent signaling pathways [6]. Some GPCR ligands preferentially activate particular signaling pathways and as such are classified as "biased" agonists. Because different signaling pathways can trigger distinct physiological outcomes, biased agonists could serve as novel therapeutics with superior functional specificity [7,8]. For example, β-arrestin-biased angiotensin II (AngII) receptor agonists promote cardioprotective signaling and contractility compared to conventional angiotensin receptor blockers but likewise antagonize deleterious G protein-mediated effects [9][10][11]. Consequently, they have been favorably evaluated for the treatment of long-term cardiomyopathy [12] in animal models and proposed as improved protectants against dysregulation of the renin-angiotensin-aldosterone system in severe COVID-19 disease [13].
Contrary to this extensive literature, several studies utilizing bioluminescence resonance energy transfer (BRET) and other experimental strategies that involve exogenous receptor overexpression indicate that SII and its derivatives lack specificity towards β-arrestin-mediated pathways and instead function as partial agonists for G protein signaling [24][25][26]. While BRET and similar approaches may facilitate direct and sensitive interrogation of the activation of different G protein isoforms [24,26], alteration of receptor expression can also introduce system bias that perturbs functional selectivity independently of ligand bias [27]. Indeed, at increased receptor densities, numerous G s -coupled receptors additionally activate noncanonical phospholipase C pathways [28]. Similarly, the adenosine A 1 receptor, which is classically G i -coupled, gains the ability to activate adenylyl cyclase through G s [29].
Accordingly, we used here a stably integrated tetracycline-inducible titratable expression system to investigate the effects of AT 1 R expression on multiple signaling responses to various agonists in a single cell line. Through selective chemical inhibition, we also assessed the roles of different G protein subfamilies in mediating these activities. Our data illuminate how receptor expression acts orthogonally of ligand bias to generate distinct functional profiles at the AT 1 R and how overexpression of receptors at levels well above those normally found in tissues can lead to erroneous conclusions about bias in physiologic contexts.
to~7.1 pmol/mg overexpression in stably transfected human embryonic kidney (HEK) 293 cells [25]. To investigate how such variations could affect signaling behaviors in the absence of confounding factors between cell lines, we sought to utilize a cellular system with consistently titratable AT 1 R expression. The creation of a stable tetracycline-inducible AT 1 R expression cell line using a TetOn system has been previously described [30]. Such systems consist of two elements-a constitutively expressed reverse tetracycline-controlled transactivator (rtTA) and a gene of interest controlled by an rtTA-dependent tetracycline response element (TRE)-and thereby enable titration of gene expression by treatment with variable amounts of doxycycline ( Fig 1A) [31]. Using whole-cell saturation binding of the radiolabeled ligands [ 3 H]-olmesartan and [ 3 H]-AngII, we measured doxycycline-induced cell surface receptor expression in a U2OS-derived cell line stably expressing a TetOn system for the AT 1 R (hereafter referred to as U2OS-TetOn-AT 1 R). Cell surface receptor density was~46 fmol/mg in the absence of doxycycline, likely due to constitutive leaky expression, but reached up to~2.6 pmol/mg at a maximal doxycycline dosage of 250 ng/ml, thus confirming a suitably broad range of receptor levels ranging from near endogenous to more than 50-fold overexpression (Fig 1B).

AT 1 R overexpression uniquely enables β-arrestin-biased agonists to initiate calcium signaling
G protein activation through the AT 1 R induces phospholipase C (PLC) to produce inositol-1,4,5-triphosphate (IP 3 ), rapidly triggering mobilization of calcium from intracellular stores [32]. Accordingly, a lack of these responses upon stimulation with SII or its derivatives is posed as evidence for these ligands' inability to activate G proteins [10,11,[14][15][16]19]. Conversely, the presence of such responses in several overexpression-based studies has been presented as evidence that these ligands are partial agonists for G protein-mediated signaling [24,25,33]. We investigated this discrepancy by profiling cytosolic Ca 2+ influx at different receptor expression levels following stimulation with each of the following ligands: unbiased AngII; the β-arrestin-biased agonists TRV023, TRV026, TRV027, and SII; or the G q/11 -biased agonist TRV055 [25,34,35]. Cells treated with AngII or TRV055 exhibited dose-dependent Ca 2+ responses at all assessed levels of receptor expression. Conversely, TRV023, TRV026, TRV027, or SII stimulation only triggered dose-dependent Ca 2+ mobilization at doxycycline-induced receptor overexpression levels of at least~1200 fmol/mg (Fig 2A and 2B). Together, these data indicate that in our inducible cell line, β-arrestin-biased AT 1 R agonists only gain the ability to initiate minimal Ca 2+ mobilization at receptor concentrations at least 25-fold higher than basal constitutive levels of receptor expression. This low expression, by contrast, is sufficient to enable unbiased and G protein-biased agonists to activate such responses.

Both G i and G q/11 mediate overexpression-exclusive calcium signaling by βarrestin-biased AT 1 R agonists
In physiological contexts, the AT 1 R primarily activates PLC and thus calcium signaling by coupling to heterotrimeric G proteins of the G q/11 family [36]. However, the AT 1 R additionally triggers some functional responses by activating G i [16,36], and Gβγ subunits released following dissociation of G i family heterotrimers can also activate PLC [37]. In agreement with this general dual G protein signaling capability of the AT 1 R, some overexpression-dependent experimental approaches find SII to activate both G q/11 and G i [24], while certain BRET biosensors even indicate a preference of SII and TRV027 to activate G i more strongly than G q/11 [26]. We therefore sought to determine the contributions of the G i and G q/11 families to the overexpression-exclusive calcium signaling induced by β-arrestin-biased AT 1 R agonists and to assess whether dependence on these G protein families differed for responses to unbiased and

PLOS ONE
Loss of biased signaling at a G protein-coupled receptor in overexpressed systems G protein-biased agonists. Pretreatment with the selective G i family inhibitor pertussis toxin (PTX) [38] partially abolished calcium responses to all β-arrestin-biased agonists but had no effect on responses to AngII or TRV055 (Fig 3A). In contrast, the selective G q/11 family inhibitor YM-254890 [39,40] fully eliminated all calcium signaling by β-arrestin-biased agonists and partially but robustly reduced responses elicited by AngII and TRV055 (Fig 3B). These data indicate that the overexpression-exclusive calcium signaling induced by β-arrestin-biased agonists is both partly mediated by G i and entirely dependent on G q/11 , implying that the two G protein families play non-additive roles in such responses and that some portion of calcium signaling depends on both G i and G q/11 . Indeed, this interpretation agrees with previously published findings that G i -mediated calcium signaling requires relief of PLC autoinhibition by active G q/11 [41] and that wholesale chemical inhibition of G q/11 activity abrogates such G idependent calcium responses [40]. Furthermore, the dependence of calcium responses to βarrestin-biased agonists on both G i and G q/11 differs sharply from the exclusive dependence of AngII and TRV055-induced calcium signaling on G q/11 . Such findings corroborate published BRET assay data describing a relative preference for G i activation over G q/11 by β-arrestinbiased agonists, albeit all at high levels of receptor expression [26].

The β-arrestin-biased AT 1 R agonist TRV026 induces receptor internalization at low receptor densities while losing functional selectivity with high receptor overexpression
In multiple cellular systems, AngII stimulation of the AT 1 R triggers functional outcomes through both G protein-and β-arrestin-2-dependent mechanisms. On the other hand, SIIinduced responses appear to be independent of G protein activation [14,[17][18][19]23], although SII and its β-arrestin-biased derivatives are less efficacious than AngII, even for β-arrestindependent responses [25,35]. Therefore, we selected a β-arrestin-biased ligand, TRV026, and examined its competency to induce receptor internalization, a process mediated by β-arrestins [5,42,43], without activating G protein in our U2OS-TetOn-AT 1 R system. We titrated AT 1 R expression to 142 fmol/mg and 230 fmol/mg, concentrations at which β-arrestin-biased agonists did not activate G protein-mediated calcium signaling (Figs 2A, 2B, 3A and 3B). Under these conditions, stimulation with both AngII and TRV026 triggered substantial receptor internalization, measured as percentage loss of cell surface receptor expression (Fig 4A). Combined with the calcium response data, these results suggest that at low receptor densities, βarrestin-biased AT 1 R agonists initiate functional responses independent of G protein activation. Such evidence recapitulates findings in vascular smooth muscle cells [19], left ventricular tissue samples [9], isolated cardiac myocytes, and Langendorff-perfused hearts [17] while contrasting with data obtained from some overexpressed receptor systems [24,26]. Additionally, agonist-induced receptor internalization percentage decreased with increasing receptor expression (Fig 4A), as would be expected [35].
At high expression levels, numerous GPCRs gain the ability to activate noncanonical effectors while also maintaining canonical pathways [28,29]. Such behavior contradicts conventional receptor theory, which postulates that all responses should increase directly and proportionally with receptor expression [44]. To examine whether a similar pattern might describe the activity of β-arrestin-biased agonists at the AT 1 R, we normalized the extent of calcium influx and receptor internalization to their respective maximum responses to enable direct comparison between these two functional outcomes at different receptor concentrations. TRV026 was effectively completely selective for the receptor internalization process over calcium signaling at low levels of receptor expression, but this preference was lost at higher receptor densities. Behaviors induced by AngII did not follow this trend; normalized calcium and internalization responses did not differ from each other at low levels of receptor expression, while calcium signaling was slightly favored over induction of receptor internalization with high receptor overexpression (Fig 4B). This apparent bias, however, is likely reflective of the differing natures of second messenger and receptor internalization assays. According to classical models of GPCR function, receptor overexpression should drive higher overall levels of G protein activation, leading to elevated or saturated readouts of second messengers. Conversely, a relative paucity of β-arrestin relative to receptor should decrease the proportion of receptor that is internalized, even though the total number of internalized receptor molecules is increased [35,45]. Together, these results indicate that at low receptor levels approaching those that are physiologically relevant, TRV026 exhibits a unique selectivity for β-arrestinmediated functional outcomes. Such a selectivity is not shared by AngII and is lost at high receptor overexpression, which drives a non-physiologic gain of function for TRV026 to activate G protein-mediated signaling.

Discussion
Biased agonism for GPCR signaling is an attractive goal for drug discovery efforts, because effecting functional outcomes with finer specificity could improve and expand therapeutic options in many disease systems [7,8,27]. β-Arrestin-biased agonism at the AT 1 R holds therapeutic potential, owing to the deleterious consequences of excessive G protein-mediated signaling and, conversely, the positive physiological effects of β-arrestin-dependent pathways through the AT 1 R [9,12,13,46,47]. Accordingly, establishing appropriate systems to test the signaling behavior of different AT 1 R ligands is important for both basic investigation of biased agonism and evaluation of potential drug candidates. Findings in overexpressed systems of G protein activation by putatively β-arrestin-biased AT 1 R agonists [24][25][26]33] raise important questions about the relevance and technical merits of different experimental strategies to profile GPCR signaling. Now, our study demonstrates that receptor overexpression markedly alters the specificity of β-arrestin-biased AT 1 R agonists, leading to gain of functions (Figs 2A,  2B, 3A, 3B and 4B) not observed in physiologically relevant systems [9,11,17,19]. These data mirror previous findings with other GPCRs and their downstream effectors [28,29], and they caution against the uncritical use of overexpression systems to characterize GPCR activity. Such systems are powerful tools for exploration and hypothesis generation, but the data they produce must be interpreted carefully and validated in more physiologically representative models.
Investigations using cell lines modified by CRISPR/Cas9-mediated genetic knockout together with application of chemical inhibition have reported that the AT 1 R and several other GPCRs fail to activate cellular signaling responses in the absence of active G proteins. Based on such results, the authors of these studies argue that GPCRs mediate downstream processes exclusively through G protein activation [48]. However, genetic deletion of important regulatory proteins is also likely to cause compensatory and inconsistent signal rewiring and is therefore an imperfect strategy to ascertain the roles of such proteins in less heavily engineered systems. Indeed, other studies have shown that the AT 1 R regulates functional responses through both G protein-dependent and independent mechanisms [14,19,23,49], the relative predominances of which may shift with changes in receptor expression. Furthermore, the system bias induced by differential expression of effectors is likely to be strongest with the high degrees of overexpression or knockdown found in engineered experimental systems, though it is even recognized that in physiological contexts, a GPCR ligand can exhibit varying functional selectivity across tissues, owing to differing expression levels of signal transducers [27]. Our own data (Fig 4A and 4B) corroborate that β-arrestin-mediated receptor internalization persists in the absence of G protein activation [48], while also further demonstrating that perturbed expression of signaling components greatly alters signaling specificity, even in the same cellular system.
What molecular mechanisms might drive perturbations in functional specificity upon receptor overexpression? Recent population-based methods such as double electron-electron resonance (DEER) spectroscopy show that receptors under various conditions are not adequately represented by singular static structures but rather constitute heterogeneous ensembles of conformational states [34]. Such conformational heterogeneity provides a potential mechanistic framework to explain changes in observable functional selectivity with receptor expression. Conformational states with a propensity to activate G protein-mediated signaling could represent a small but nonzero proportion of β-arrestin-biased agonist-bound AT 1 R ensembles. At near-physiologic levels of receptor expression, this population would contribute negligibly to the overall cellular signaling response, leading to a lack of G protein activation. By contrast, overexpression would increase the total number of β-arrestin-biased agonistbound receptor molecules that adopt these G protein-favoring conformational states, thereby triggering measurable amounts of G protein activation. These "numbers game" mechanisms are also compatible with the ability of β-arrestins to uncouple or desensitize G protein-mediated signaling [3,4]; overexpression of the receptor combined with saturating dosing could allow the number of β-arrestin-biased agonist-bound AT 1 R complexes to exceed the endogenous population of β-arrestins, leading to appreciable G protein activation that would be fully suppressed under physiological conditions. Indeed, in similar inducible expression systems, βarrestin overexpression counteracts increases in G protein-mediated responses following receptor overexpression [30].
Indeed, in our study, a concentration of the G q/11 family inhibitor YM-254890 that fully abolishes calcium responses to β-arrestin-biased agonists at all AT 1 R expression levels and to AngII and TRV055 at low receptor levels only partially eliminates calcium signals induced by the latter group of agonists with high receptor overexpression (Fig 3B). These results imply that for a given receptor concentration, the number of total AT 1 R molecules capable to activate G protein is much higher under stimulation with unbiased or G protein-biased agonists than with β-arrestin-biased agonists. Thus, at high receptor levels, unbiased and G protein-biased agonists at their saturating concentrations activate G protein too robustly to be completely abolished with YM-254890 at the concentration utilized in our study. As a further possibility for the partial inhibitory effect of YM-254890 at the saturating concentrations of unbiased or G protein-biased agonists, the AT 1 R has been reported to signal additionally through G 12/13 [26] and possibly G s . The activation of these noncanonical G proteins by the AT 1 R could theoretically initiate calcium signaling either through free G protein subunit activity or production of second messengers. Such activities remain uncharacterized.
We conclude that receptor overexpression can cause signaling outcomes to diverge from those observed in physiologically relevant systems. This results in loss of ligand bias observed at more physiological levels of receptor expression and presents an important obstacle to drug development unless more widely appreciated.

Cell culture
U2OS-TetOn-AT 1 R cells were generated as previously described [30]. Briefly, U2OS cells were transfected with pTet-On and subjected to selection with 500 μg/ml G418 for 3 weeks. Stable clones were then transfected with pTRE2-hyg-HA-AT 1 R and subjected to selection with 200 μg/ml hygromycin for 3 weeks. Positive clones with low background and ability to express AT 1 R upon doxycycline treatment were maintained in minimal essential medium with 100 μg/μl G418 and 100 μg/μl hygromycin supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 mM GlutaMAX (Gibco).

Radioligand binding and receptor internalization measurement
U2OS-TetOn-AT 1 R cells grown at~165,000 cells/well in poly-D-lysine-coated 12-well plates were treated with doxycycline for 14 h. For receptor expression measurements, media was removed from plates and the cells were incubated in minimal essential medium containing 20 nM [ 3 H]-AngII or 50 nM [ 3 H]-olmesartan for 1.5 h. Nonspecific binding was measured by simultaneous addition of 200 μM candesartan to radioligand-containing media. Cells were washed with ice-cold PBS containing MgCl 2 and CaCl 2 and lysed with a solution of 0.1% SDS and 0.5 N NaOH. Radioactivity of cell lysates was measured by scintillation counting. Total protein was determined using the Pierce BCA protein assay (Thermo Scientific) and used to convert scintillation counts to cell surface AT 1 R concentration in Radioactivity Calculator (GraphPad Software). The full protocol for these experiments is publicly archived at dx.doi. org/10.17504/protocols.io.ewov1oxe2lr2/v1. For receptor internalization measurements, experiments were performed as above, except that cells were stimulated with unlabeled ligand for 1 hr and washed with an ice-cold acid stripping solution consisting of 0.2 M acetic acid and 0.5 M NaCl adjusted to pH 2.5 for 5 min prior to addition of radiolabeled ligand, and [ 3 H]-AngII was used for all measurements. The extent of ligand-induced receptor internalization was determined as loss of cell surface receptor expression upon stimulation with ligands. The full protocol for these experiments is publicly archived at dx.doi.org/10.17504/protocols.io. x54v9dz91g3e/v1.

Calcium fluorimetry
U2OS-TetOn-AT 1 R cells grown at 15,000 cells/well in poly-D-lysine-coated 96-well black well plates were treated with doxycycline for 14 h. For studies of PTX inhibition of calcium signaling, cells were treated with 100 ng/ml PTX or vehicle concurrent with addition of doxycycline. Media was removed from plates and the cells were incubated with calcium-sensitive fluorescent dye from the FLIPR Calcium 6 assay kit for 2 h according to the manufacturer's instructions. For studies of YM-254890 inhibition of calcium signaling, cells were treated with 1 μM YM-254890 or vehicle for 1 h. Fluorescence was measured in a FlexStation 3 microplate reader (Molecular Devices) for 3 min according to the manufacturer's instructions. Cells were stimulated with ligand 20 s after starting fluorescence measurements. Fluorescence measurements for each well were adjusted to baseline by subtracting the average signal from the first 10 s of measurement. All calcium responses were quantified as baseline-adjusted peak fluorescent signal. The full protocol for these experiments is publicly archived at dx.doi.org/10.17504/ protocols.io.3byl4jdmjlo5/v1.

Graphing and statistical analysis
Raw calcium assay data were analyzed and graphed using MATLAB R2020b (Mathworks). All other data were graphed using Prism 9.4 (GraphPad Software). Calcium data were fitted to a log(agonist) versus response model with three parameters (baseline, span, and EC 50 ) in Prism 9.4. Statistical analyses were also performed in Prism 9.4. Calcium dose response data were subjected to ordinary two-way analysis of variance (ANOVA) and post-hoc multiple comparisons tests of peptide agonist dose effects within groups of different receptor expression levels. A statistically significant difference of calcium response values between at least one agonist dosage and the minimum tested dose, as assessed by Dunnett's multiple comparisons tests, was interpreted to indicate a dose-dependent response. The effects of PTX and YM-254890 treatment were analyzed using ordinary two-way analysis of variance (ANOVA) and post-hoc Šidák's multiple comparisons tests of inhibitor effects within groups of different receptor concentrations. Receptor internalization responses were subjected to one-sample two-tailed t-tests comparing each experimental group to a null hypothesis mean of zero percent internalization, with significance interpreted to indicate ligand-induced internalization. To enable statistical testing between different functional readouts, calcium and internalization responses were normalized, per ligand, to percent of the maximal mean response in their respective assay format across all receptor levels. They were then compared at different receptor concentrations using two-way repeated measures ANOVA and post-hoc Šidák's multiple comparisons tests at each tested receptor expression level.